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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
  • C12N15/1132—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses against retroviridae, e.g. HIV
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  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/18—Type of nucleic acid acting by a non-sequence specific mechanism
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/30—Chemical structure
  • C12N2310/31—Chemical structure of the backbone
  • C12N2310/315—Phosphorothioates

Definitions

• the present invention relates to an anti-HIV (Human Immunodeficiency Virus) agent containing as an active ingredient a phosphodiester bonding type or phosphorothioester bonding type oligoribonucleotide which shows an excellent anti-viral action for diseases caused by HIV.
• HIV Human Immunodeficiency Virus
• the inventors made an extensive research on obtaining a low molecular oligonucleotide with the activity for inhibition of HIV replication, and recently found that a short chain oligodeoxyguanylic acid of 4-8 bases had the activity for inhibition of HIV replication.
• the invention provides an anti-HIV agent characterized in that it comprises as an active ingredient one or more species selected from phosphodiester bonding type oligoribonucleotides or phosphorothioester bonding type oligoribonucleotides represented by the below formulae (I)-(XXIII) described below in the item's a)-e).
• the oligoribonucleotide of the above formulae (I)-(XXIII) which is an active ingredient of the anti-HIV agent of the invention can generally be synthesized by usual methods (see Scaringe et al., Nucleic Acids Res. 18 , 5433; Damha and Ogilvie, Methods in Mol. Biol. 20 81; Eckstein, Ann. Rev. Biochem. 54 , 367).
• a favorable and representative synthetic method as a synthetic method for the compounds of the invention is illustrated as [Synthetic method A] - [Synthetic method E] in the followings.
• the amidite reagent (5'-o-dimethoxytrityl)-2'-o-(t-butyldimethylsilyl)-ribonucleoside-3'-N, N-diisopropyl(cyanoethyl)-phosphoramidite, is subjected to the condensation by tetrazole in which the amino group in guanylic acid base as a monomer is protected by isobutyl group. This is followed by the oxidation of the phosphorous part with iodine to lead a phosphoric acid triester derivative. The above reaction is repeated until an aimed chain length is obtained.
• An ammonia treatment then converts each internucleotide from the triester to the diester and carries out the detachment from the support and the simultaneous deblocking of the base part. This is followed by a tetra-N-butylammonium fluoride treatment to deblock 2'-hydroxyl protecting group giving an aimed oligoribonucleotide phosphorylated at the 3'-terminal position in this method.
• the amidite reagent (5'-o-dimethoxytrityl)-2'-o-(t-butyldimethylsilyl) ribonucleoside-3'-N, N-diisopropyl (cyanoethyl)phosphoramidite, is subjected to the condensation by tetrazole in which the amino group of guanylic acid base as a monomer is protected by isobutyl group.
• the unreacted 5'-hydroxyl group is acetylated by acetic anhydride and N-methylimidazole, etc.
• the amidite reagent (5'-o-dimethoxytrityl)-2'-o-(t-butyldimethylsilyl) ribonucleoside-3'-N,N-diisopropyl (cyanoethyl) phosphoramidite, is subjected to the condensation by tetrazole in which the amino group in guanylic acid base as a monomer is protected by isobutyl group. This is followed by the oxidation of the phosphorous part with iodine to lead to a phosphoric acid triester derivative.
• the 5' protecting group (DMTr) of this guanylic acid is then removed by trichloroacetic acid, and as above is condensed the amidite reagent in which functional groups of the second guanylic acid are protected. This is followed by the sulfiding of the second phosphorous part with tetraethylthiuram disulfide to lead to a phosphorothiotriester. After the condensation of guanylic acid and the sulfiding of the phosphorous part are repeated until an aimed chain length is obtained, an ammonia treatment converts each internucleotide from the triester to the diester and carries out the detachment from the support and the simultaneous deblocking of the base part.
• DMTr 5' protecting group
• the phosphoramidite method is explained in case of obtaining oligoribonucleotide with a phosphorothioester bonding.
• a material in which a functionally protected guanosine is bound to a support such as controlled-pore glass (CPG), is prepared. After the protective 4,4'-dimethoxytrityl group of this nucleoside's 5'-hydroxyl is deprotected by trichloroacetic acid, etc.
• the amidite reagent (5'-o-dimethoxytrityl-2')-o-(t-butyldimethylsilyl) ribonucleoside-3'-N, N-diisopropyl (cyanoethyl) phosphoramidite, is subjected to the condensation by tetrazole in which the amino group of guanylic acid base is protected by isobutyl group.
• the unreacted 5'-hydroxyl group is acetylated by acetic anhydride and N-methylimidazole, etc.
• the amidite reagent (5'-o-dimethoxytrityl)-2'-o-(t-butyldimethylsilyl) ribonucleoside-3'-N, N-diisopropyl(cyanoethyl)phosphoramidite, is subjected to the condensation by tetrazole in which the amino group of guanylic acid base is protected by isobutyl group.
• the unreacted 5'-hydroxyl group is acetylated by acetic anhydride and N-methylimidazole, etc.
• HIV Anti-AIDS virus
• MT-4 cells (6 x 10 4 cells per well).
• the cells were then infected with AIDS virus [HIV-1(IIIB) : ca. 50 TCID 50 (50% tissue culture infectious doses)/well] and were incubated in a 5% CO 2 atmosphere at 37°C for 5 days.
• HIV-1(IIIB) ca. 50 TCID 50 (50% tissue culture infectious doses)/well
• viable cells were measured by MTT method after 5 days' incubation. The results are shown in Table 1.
• each oligoribonucleotide in the table the meaning of p and s is identical to the definition described above, and G, A, C and U are guanosine, adenosine, cytidine and uridine respectively.
• the phosphodiester bonding type or phosphorothioester bonding type oligoribonucleotides which are the active ingredients of the anti-HIV agents of the invention showed the effect even from 2 bases, and in 3-8 bases the excellent anti-HIV activity was shown. Further, no cytotoxicity was shown at the concentration of 100 ⁇ g/ml in any oligoribonucleotide.
• a phosphodiester bonding type oligoguanylic acid whose terminal G is not phosphorylated had no effect in 2 or 3 bases and showed a little effect in 4 bases.
• novel anti-HIV agents of the invention are useful in particular as a medicament for treating diseases infected by a retrovirus such as AIDS, and can be processed with pharmaceutical vehicles to oral preparations such as tablets, capsules, granules, fine granules and powders, or to non-oral preparations such as injections, i.v. drip infusions and suppositories.
• oral preparations such as tablets, capsules, granules, fine granules and powders
• non-oral preparations such as injections, i.v. drip infusions and suppositories.
• oral preparations are, for example, tablets, capsules, granules, fine granules and powders, and those of the non-oral preparations are injections and suppositories.
• the required dose as oral preparations depends on the age and body weight of each patient and the severity of the disease, though, the daily dose for adults is usually between 0.1-6 g which are favorably administered in several times.
• oral preparations such as the above tablets, capsules and granules can be prepared by the conventional method using, for example, starch, lactose, sugar, mannite, carboxymethylcellulose or inorganic salts.
• Starch dextrin, Arabic gum powder, gelatin, hydroxypropylstarch, methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose, crystalline cellulose, ethylcellulose, polyvinylcellulose, macrogol.
• Starch hydroxypropylstarch, sodium carboxymethylcellulose, calcium carboxymethylcellulose, carboxymethylcellulose, low substituted hydroxypropylcellulose.
• Talc wax, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, polyethylene glycol.
• oligoribonucleotides of the formulae (I) - (XXIII) of the invention can be administered as suspensions, liquid emulsions, syrups or elixirs.
• flavorings or colorings can be included.
• the required dose as non-oral preparations depends on the age and body weight of each patient and the severity of the disease, though, the daily dose of the above oligoribonucleotides for adults is usually between 1-100mg which are administered by intravenous injection, i.v. drip infusion, subcutaneous injection or intramuscular injection.
• non-oral preparations can be prepared by the conventional method.
• diluents can generally be used distilled water for injection, physiological saline solution, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol or the like.
• the preparations can contain antiseptics, preservatives or stabilizers as required.
• the non-oral preparations are filled into vials or, etc., and frozen and dried by the conventional lyophilizing technology.
• Liquid preparations can be prepared again just before use by using this lyophilizates. Further, there may be appropriately added isotonic agents, stabilizers, preservatives, soothing agents or, etc. , as required.
• embrocations such as externally applicable liquids and ointments, and suppositories for rectal administration can be illustrated and all these can be prepared by a conventional method.
• Formulation 1 Crystalline cellulose 34.5 wt. parts 2 10% ethanolic hydroxypropylcellulose solution 50 wt. parts 3 Calcium carboxymethylcellulose 5 wt. parts 4 Magnesium stearate 0.5 wt. parts 5 pGpGpGpG 10 wt. parts Total 100 wt. parts
• novel anti-HIV agents of the invention are effective for the treatment of the HIV (AIDS virus) induced diseases such as AIDS and ARC.

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• 1995
  • 1995-07-21 EP EP95926009A patent/EP0724882A4/fr not_active Withdrawn

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